Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect

Extraluminal factors contributing to inflammatory bowel disease

Many identified and yet unknown factors contribute to the pathogenesis of inflammatory bowel disease (IBD). The genome-wide association studies clearly support the earlier developed concept that IBD occurs in genetically predisposed individuals who are exposed to distinct environmental factors, which together result in dysregulation of the mucosal immune system. Thus, the majority of previous studies have focused on the immune response within the intestinal wall. The present review aims to emphasize the contribution of three extraluminal structures to this inflammatory process, namely the mesenteric fat tissue, the lymphatics and the microvasculature. Broadening our view across the intestinal wall will not only facilitate our understanding of the disease, but will also us to identify future therapeutic targets

Continuous Glucose Monitoring and Physical Activity

Continuous glucose monitoring (CGM) use has several potential positive effects on diabetes management. These benefits are, e.g., increased time in range (TIR), optimized therapy, and developed documentation. Physical activity is a recommended intervention tool in diabetes management, especially for people with type 2 diabetes (T2D). The benefits of physical activity for people with diabetes can be seen as an improvement of glycemic control, glycemic variability, and the reduction of insulin resistance. In relation to the physical activity of people with T2D, the benefits of CGM use can even be increased, and CGM can be a helpful tool to prevent adverse events due to physical activity of people with diabetes, such as hypoglycemic events and nocturnal hypoglycemia after sports. This narrative review aims to provide solid recommendations for the use of CGM in everyday life physical activities based on the noted benefits and to give a general overview of the guidelines on physical activity and CGM use for people with diabetes

Comparison of conventional ROTEM® cups and pins to the ROTEM® cup and pin mini measuring cells (MiniCup)

BACKGROUND: Thromboelastometry (ROTEM®) is a well-established measurement to guide perioperative coagulation management. Recently, an enhancement for low volume specimens using cup and pin mini measuring cells (MiniCup) was invented for scientific non-clinical use. The MiniCup measuring cells allow testing with half of the blood volume required to run a test as compared to the conventional ROTEM® cup and pin (150 μL vs. 300 μL per assay). OBJECTIVES: The aim of this prospective study was to compare ROTEM® results using conventional cups and pins to the results of the MiniCup measuring cells. METHODS: Blood samples drawn during major pediatric surgery were analyzed using the conventional ROTEM® and the MiniCup system to compare parameters from the EXTEM, INTEM and FIBTEM assays. RESULTS: Citrated blood samples (n = 120) from 70 pediatric patients were analyzed. Results of the MiniCup cells were considerably different than the conventional ROTEM® cups and pins measurements. The MiniCup results show less clot firmness and demonstrate higher variability in the parameters reflecting the kinetics of clot building. CONCLUSION: The MiniCup measuring cells may offer advantages in pediatric care or research facilities, but specific reference ranges need to be established first, and adequate reproducibility must be determined by further studies before clinical use can be recommended

The IKK inhibitor Bay 11-7082 induces cell death independent from inhibition of activation of NFκB transcription factors.

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells